Novel Strain of Lactobacillus Paracasei Subspecies Paracasei Having Antimicrobial and Immunomodulatory Properties

ABSTRACT

The invention relates to a novel strain of  Lactobacillus paracasei  subspecies  paracasei , having antimicrobial and immunomodulatory properties, and to compositions containing said strain.

The invention relates to a novel strain of Lactobacillus paracasei subsp. paracasei having antimicrobial and immunomodulatory properties.

A very large number of scientific studies have reported the beneficial effects, on the health, of certain microorganisms present in fermented foodstuffs, in particular dairy products. These microorganisms are commonly referred to as “probiotics”. According to the definition generally accepted at the current time, probiotics are: “live microorganisms which, when they are consumed in appropriate amounts, have a beneficial effect on the health of the host” (FAO/WHO report on evaluation of health and nutritional properties of probiotics in food, including powder milk containing live lactic acid bacteria; Cordoba, Argentina; Oct. 1-4, 2001).

It has been shown that the consumption of food products containing probiotic bacteria can produce favorable effects on the health, in particular through re-equilibrating the intestinal flora, improving resistance to infections, and modulating the immune response.

The probiotic microorganisms used in human food are generally lactic acid bacteria belonging mainly to the Lactobacillus and Bifidobacterium genera, and in particular to the species Lactobacillus paracasei subsp. paracasei (a strain of which is described in patent application EP0794707).

However, the beneficial effects on the health are not generally common to all the bacteria of the same genus, nor even of the same species. They are, most commonly, encountered only in certain strains; in addition, the effects observed can vary qualitatively and/or quantitatively from one probiotic strain to the other, including within the same species.

In order for it to be possible for a microorganism to be considered potentially usable as a probiotic, it must meet at least one, and ideally several, of the following criteria:

-   -   exhibit an inhibitory activity with respect to pathogenic         microorganisms that may be present in the intestinal flora, it         being possible for this activity to result either from the         ability to adhere to the intestinal cells, thus excluding or         reducing the adherence of the pathogens, or from the ability to         produce substances which inhibit the pathogens, or from the         combination of these two characteristics;     -   exhibit immunomodulatory properties, and in particular         immunostimulatory and/or anti-inflammatory properties.

In addition, if this microorganism is intended to be incorporated into a dairy product, it should preferably exhibit satisfactory growth on milk.

Finally, it should maintain good viability, during the production and storage of the foodstuff into which it is incorporated, and also after ingestion of this foodstuff by the consumer, so as to be able to reach the intestine and to survive in the intestinal environment.

It should, however, be noted that, although viability is essential in order to correspond to the current definition of “probiotic”, it has been shown that some of the beneficial effects associated with probiotic strains can be obtained even in the absence of live bacteria, and are attributable to certain bacterial fractions or to active fractions of their culture supernatants. For example, PCT application WO2004093898 describes an immunomodulatory preparation obtained by fractionation of the culture supernatant of the CNCM I-2219 strain.

The inventors have now succeeded in isolating a novel strain of Lactobacillus paracasei subsp. paracasei which meets the criteria indicated above.

A subject of the present invention is this strain, which was deposited, according to the Treaty of Budapest, with the CNCM (Collection Nationale de Cultures de Microorganismes [National collection of microorganism cultures], 25 rue du Docteur Roux, Paris), on Nov. 9, 2006, under number I-3689.

The CNCM I-3689 strain has the following characteristics:

-   -   Morphology: Gram-positive microorganism, small thin bacilli,         isolated or small chains.     -   Fermentation of the following sugars (results obtained on an api         50 CH strip-API MRS medium at 37° C. for 48 h): ribose,         galactose, D-glucose, D-fructose, D-mannose, mannitol,         N-acetylglucosamine, arbutin, cellobiose, maltose, lactose,         trehalose, melezitose, D-turanose, D-tagatose, gluconate.     -   Presence of a single CRISPR locus of sequence SEQ ID NO: 1,         containing a repeat sequence represented by the nucleotide         sequence SEQ ID NO: 2.

It has, in addition, antimicrobial properties which result in a strong ability to inhibit the growth of pathogenic microorganisms in culture, in particular Escherichia coli, Salmonella enteritidis and Listeria monocytogenes.

The CNCM I-3689 strain also has immunomodulatory, and in particular anti-inflammatory, properties.

The subject of the present invention also encompasses Lactobacillus paracasei subsp. paracasei strains that can be obtained by mutagenesis or by genetic transformation of the CNCM I-3689 strain. Preferably, these strains retain the antimicrobial and immunomodulatory properties of the CNCM I-3689 strain. They may be strains in which one or more of the endogenous genes of the CNCM I-3689 strain has (have) been mutated, for example so as to modify some of its metabolic properties (e.g. the ability of this strain to metabolize sugars, its resistance to intestinal transit, its resistance to acidity, its post-acidification or its metabolite production). They may also be strains resulting from genetic transformation of the CNCM I-3689 strain with one or more gene(s) of interest, making it possible, for example, to confer additional physiological characteristics on said strain, or to express proteins of therapeutic or vaccine interest, which it is desired to administer by means of said strain.

These strains can be obtained from the CNCM I-3689 strain by means of the conventional techniques for random or site-directed mutagenesis and genetic transformation of lactobacilli, such as those described, for example, by Gury et al. (Arch Microbiol., 182, 337-45, 2004) or by Velez et al. (Appl Environ Microbiol., 73, 3595-3604, 2007), or by the technique known as “genome shuffling” (Patnaik et al. Nat Biotechnol, 20, 707-12, 2002; Wang Y. et al., J. Biotechnol., 129, 510-15, 2007). These strains have in particular a CRISPR locus of sequence SEQ ID NO: 1.

A subject of the present invention is also a method for obtaining a Lactobacillus paracasei subsp. paracasei strain having antimicrobial and/or immunomodulatory properties, comprising a step of mutagenesis or of genetic transformation of the CNCM I-3689 strain.

A subject of the present invention is also a method for obtaining a cell fraction having antimicrobial and/or immunomodulatory properties, from a Lactobacillus paracasei subsp. paracasei strain in accordance with the invention. Said cell fractions are in particular DNA preparations or bacterial wall preparations obtained from cultures of said strain. They may also be culture supernatants or fractions of these supernatants.

A subject of the present invention is also compositions comprising a Lactobacillus paracasei subsp. paracasei strain in accordance with the invention, or a cell fraction obtained from said strain.

These compositions can in particular be lactic ferments, combining a Lactobacillus paracasei subsp. paracasei strain in accordance with the invention with one or more other, optionally probiotic, strain(s) of lactic acid bacteria. By way of example of strains of lactic acid bacteria, mention may be made of the Lactobacillus bulgaricus and Streptococcus thermophiles strains.

They may also be food products, and in particular dairy products, or pharmaceutical or cosmetic products comprising a Lactobacillus paracasei subsp. paracasei strain in accordance with the invention, or a cell fraction obtained from said strain.

When said strain is present in the form of live bacteria, they will preferably be present in a proportion of at least 10⁵ cfu per gram, advantageously at least 10⁶ cfu per gram of product, more advantageously at least 10⁷ cfu per gram, and even more advantageously at least 10⁸ cfu per gram.

The present invention will be understood more clearly from the further description which follows, which refers to examples illustrating the antimicrobial, immunomodulatory and anti-infective properties of the CNCM I-3689 strain, and also the molecular typing of this strain.

EXAMPLE 1 Comparison of the Properties of the CNCM I-3689 Strain with Those of Known Probiotic Strains

The properties of the CNCM I-3689 strain were compared with those of various prior art strains, known for their probiotic properties.

The list of these strains is given in Table I below.

TABLE I Publication number of patent Genus Species Name(s) applications Lactobacillus johnsonii NCC533 = La1 = EP0577903 CNCM I-1225 Lactobacillus acidophilus NCFM WO2004032639 Lactobacillus acidophilus La-5 Lactobacillus casei Shirota Lactobacillus paracasei CRL431 = ATCC 55544 Lactobacillus rhamnosus LGG = U.S. Pat. No. ATCC 53103 4,839,281 Lactobacillus rhamnosus HN001 = WO9910476 NM97/09514 Lactobacillus plantarum ATCC 14917 = DSMZ 20174 = WCFS1 Lactobacillus plantarum Probi 299v = WO9391823 DSM 9843 Lactobacillus reuteri DSMZ 20016 Lactobacillus reuteri Biogaia SD 2112 = WO2004034808 ATCC 55730 Bifidobacterium breve ATCC 15700 Bifidobacterium breve BBC50 = EP1189517 CNCM I-2219 Bifidobacterium infantis ATCC 15697 Bifidobacterium longum BB536 Bifidobacterium longum NCC2705 = CNCM I-2618 Bifidobacterium animalis BB12 = ATCC 27536 subsp. lactis Bifidobacterium animalis HN019 = NM97/01925 WO9910476 subsp. lactis

1—Antimicrobial Activity

The investigation of antimicrobial activities was carried out against three target pathogenic bacteria: Escherichia coli E1392-75-2A, Salmonella enteritidis NIZO B1241 and Listeria monocytogenes 4B. The lactic acid bacteria were cultured on Petri dishes in two different media: Elliker medium (Elliker et al., J. Dairy Sci., 39, 1611-1612, 1956), and TGE medium (tryptone-glucose-meat extract).

The dishes are incubated at 37° C. until bacteria colonies appear. The Bifidobacterium cultures were carried out under anaerobic conditions. A layer of agar containing BHI (brain-heart infusion) medium and the pathogen is then poured at the surface of the dishes. The dishes are incubated again at 37° C., for 24 h. The diameters of the areas of pathogen inhibition are then measured around each colony of lactic acid bacteria. Score 1 corresponds to a diameter of between 1 and 3 mm. Score 2 corresponds to a diameter of between 4 and 6 mm. Score 3 corresponds to a diameter of greater than 6 mm. Each experiment was carried out three times independently for each strain.

The scores obtained on the target pathogens in each experiment were added, so as to obtain, for each lactic acid bacterium, an overall score for antimicrobial activity.

The results are given in Table II hereinafter.

These results show that, among the strains tested, the CNCM I-3689 strain is, with the ATCC 55544 strain, the one which has the highest antimicrobial activity.

TABLE II (1) (2) (3) (4) (5) Anti-pathogen E. coli/ Listeria/ Listeria/ Salmonella/ Salmonella/ score Genus Species Reference Elliker Elliker TGE Elliker TGE (1 + 2 + 3 + 4 + 5) Lactobacillus paracasei DN 114121 = 3 3 2 3 2 13 subsp. CNCM I-3689 paracasei Lactobacillus paracasei CRL431 = 2 3 3 3 2 13 ATCC 55544 Lactobacillus rhamnosus LGG = ATCC 53103 2 1 2 3 3 11 Lactobacillus plantarum ATCC 14917 = 2 1 2 3 2 10 DSMZ 20174 = WCFS1 Lactobacillus casei Shirota 2 3 1 1 2 9 Lactobacillus rhamnosus HN001 = NM97/09514 1 1 2 3 2 9 Lactobacillus johnsonii NCC533 = 1 1 1 2 1 6 LA1 = I-1225 Lactobacillus plantarum Probi 299v = 1 1 1 1 1 5 DSM 9843 Lactobacillus acidophilus La-5 1 1 2 0 1 5 Lactobacillus acidophilus NCFM 1 1 1 0 1 4 Bifidobacterium animalis BB12 = ATCC 27536 1 0 0 0 2 3 subsp. lactis Lactobacillus reuteri Biogaia SD 2112 0 1 0 1 0 2 ATCC 55730 Bifidobacterium longum BB536 0 0 0 0 0 0 Bifidobacterium animalis HN019 = NM97/01925 0 0 0 0 0 0 subsp. lactis Lactobacillus reuteri DSMZ 20016 0 0 0 0 0 0 Bifidobacterium infantis ATCC 15697 0 0 0 0 0 0 Bifidobacterium breve BBC50 = I-2219 0 0 0 0 0 0 Bifidobacterium breve ATCC 15700 0 0 0 0 0 0 Bifidobacterium longum NCC2705 = 0 0 0 0 0 0 CNCM I-2618

2—Immunomodulation

The immunomodulatory properties of the various lactic acid bacteria were evaluated by measuring the IL-10/IL-12 ratio.

Human blood samples, obtained from healthy individuals, were diluted to a 1:2 ratio with PBS-CA (Gibco) and purified using a Ficoll (Gibco) gradient. After centrifugation at 400×g for 30 min at 20° C., the PBMCs (peripheral blood mononuclear cells) were taken. Three washing steps were then carried out, and then the PBMCs were resuspended in an RPMI culture medium (Gibco) supplemented with 1% of fetal calf serum, 1% of L-glutamine (Gibco) and 150 μg/ml of gentamicin (Gibco). The PBMCs were counted under a microscope and adjusted to a concentration of 2×10⁶ cells/ml, and then distributed, in aliquots of 1 ml, into 24-well cell culture plates (Corning, Inc.).

The Lactobacillus strains were cultured in MRS medium (de Man et al., J. Appl. Bacteriol. 23, 130-135, 1960), and the Bifidobacterium strains were cultured in MRS medium supplemented with 0.03% of L-cysteine (Sigma) under anaerobic conditions. All the strains were incubated at a temperature of 37° C. The growth of the bacteria was stopped in the stationary phase, and the bacteria were then washed and resuspended at a concentration of 3 MacFarlan units in PBS containing 20% glycerol.

A volume of 10 μl of bacterial preparation was added to each well of the plates containing the PBMCs (bacteria:cell ratio 10:1). The plates were incubated for 24 h at 37° C. under an atmosphere containing 5% CO₂. The supernatant was then drawn up, centrifuged at 2000 rpm and stored at −20° C.

The control bacterial strains, with known immunomodulatory properties, were included in the test. PBS-20% glycerol, without bacteria, was also used as a negative control. The experiment was carried out for each strain on PBMCs derived from three different donors.

The expression of the cytokines was measured by means of ELISA assays using commercial kits (Pharmingen, BD Biosciences). Two cytokines were studied: IL-10 and IL-12.

For each strain tested, the average of the IL-10/IL-12 ratio was calculated. These results are given in Table III below.

TABLE III IL-10/IL-12 Genus Species Reference ratio Lactobacillus paracasei subsp. DN 114121 = 11.8 paracasei CNCM I-3689 Lactobacillus plantarum Probi 299v = 12.7 DSM 9843 Lactobacillus acidophilus La-5 23.5 Lactobacillus acidophilus NCFM 23.6 Lactobacillus johnsonii NCC533 = 23.7 La1 = I-1225 Lactobacillus plantarum ATCC 14917 = 25.0 DSMZ 20174 = WCFS1 Lactobacillus reuteri Biogaia SD 2112 25.4 ATCC 55730 Bifidobacterium longum BB536 26.4 Bifidobacterium animalis subsp. HN019 = 28.7 lactis NM97/01925 Bifidobacterium animalis subsp. BB12 = 32.4 lactis ATCC 27536 Lactobacillus rhamnosus LGG = ATCC 53013 34.5 Lactobacillus reuteri DSMZ 20016 42.0 Bifidobacterium infantis ATCC 15697 51.4 Bifidobacterium breve BBC50 = 1-2219 58.6 Lactobacillus casei Shirota 67.6 Bifidobacterium breve ATCC 15700 68.5 Bifidobacterium longum NCC2705 = 72.6 CNCM I-2618 Lactobacillus rhamnosus HN001 = 92.1 NM97/09514 Lactobacillus paracasei CRL431 = 164.0 ATCC 55544

These results show that the CNCM I-3689 strain exhibits considerable anti-inflammatory properties on this model. None of the reference strains tested exhibits such good properties.

FIG. 1 represents the IL-10/IL-12 ratio of each bacterial strain as a function of its anti-pathogen score (arbitrary unit) determined above. This figure shows how much the CNCM I-3689 strain differs from the other strains tested.

3—Survival with Respect to Intestinal Stress

An in vitro model reflecting the conditions of intestinal stress was used.

Cultures of lactic acid bacteria are prepared in milk supplemented with yeast extract. The cultures are incubated for 24 to 48 h, depending on the species (until the stationary phase of the culture).

An artificial intestinal juice composed of porcine bile salts (at 3.3 g/l) and of NaHCO₃ carbonate buffer (at 16.5 g/l) is prepared. The pH is adjusted to 6.3. 1 ml of this intestinal juice is added to 100 μl of bacterial culture. The cultures are then incubated for 5 hours. Next, the bacterial populations before and after the stress are evaluated on dishes.

The values are expressed in the following way:

intestinal stress=log(cfu5h/cfu0h).

cfuXmin being the concentration of bacteria expressed as Colony Forming Units (CFUs) after X minutes of incubation.

For the intestinal stress, survival is good when the value is greater than −0.5, moderately good when the value is between −0.5 and −1.5, and poor when the value is less than −1.5.

The results are given in the following Table IV.

TABLE IV Intestinal Genus Species Reference stress Lactobacillus paracasei subsp. DN 114121 = −0.40 paracasei CNCM I-3689 Bifidobacterium animalis subsp. HN019 = 3.40 lactis NM97/01925 Bifidobacterium infantis ATCC 15697 2.79 Lactobacillus reuteri Biogaia SD 2112 1.00 ATCC 55730 Lactobacillus johnsonii NCC533 = La1 = 0.69 I-1225 Bifidobacterium animalis subsp. BB12 = ATCC 0.07 lactis 27536 Lactobacillus paracasei CRL431 = ATCC 0.04 55544 Lactobacillus rhamnosus HN001 = 0.00 NM97/09514 Lactobacillus rhamnosus LGG = ATCC 53103 −0.07 Bifidobacterium breve ATCC 15700 −0.14 Bifidobacterium longum NCC2705 = -0.22 CNCM I-2618 Lactobacillus acidophilus La-5 −0.23 Lactobacillus plantarum ATCC 14917 = −0.79 DSMZ 20174 = WCFS1 Bifidobacterium breve BBC50 = I-2219 −0.91 Lactobacillus plantarum Probi 299v = −1.04 DSM 9843 Bifidobacterium longum BB536 −1.12 Lactobacillus acidophilus NCFM −1.45 Lactobacillus casei Shirota −1.50 Lactobacillus reuteri DSMZ 20016 −1.91

4—Conclusion

The results illustrated in Tables II, III and IV above show that, among the various strains tested, the CNCM I-3689 strain is the only one to have both considerable antimicrobial properties and considerable anti-inflammatory properties, accompanied, in addition, by very good properties of resistance to intestinal stress.

EXAMPLE 2 Growth of the CNCM I-3689 Strain on Milk

The growth-on-milk properties of the CNCM I-3689 strain were tested using the following protocol:

-   -   A medium made up of skimmed milk reconstituted with water to         which skimmed milk powder has been added was inoculated with the         CNCM I-3689 strain (5.6×10⁶ cfu/g or 1.1×10⁷ cfu/g).     -   The fermentative activity of this strain, which is linked to its         growth, is measured by continuously monitoring the pH of the         growth medium.

The results are illustrated by the graph shown in FIG. 2.

These results show that the CNCM I-3689 strain is capable of growing efficiently on milk, and that it can therefore be used in the manufacture of fermented dairy products.

EXAMPLE 3 Molecular Typing of the CNCM I-3689 Strain

Many prokaryotic organisms have one or more CRISPR loci

-   -   acronym for “Clustered Regularly Interspaced Short Palindromic         Repeats” (Jansen et al., 2002, OMICS, Vol. 6, No. 1, 23-33). A         CRISPR locus is characterized by noncontiguous repeat sequences         (or DRs), generally of 21 to 37 base pairs (bp), separated by         unique sequences, generally of 20 to 40 base pairs, called         variable sequences (“spacers”). From one bacterial strain to the         other, it is possible to observe differences regarding:     -   the number of CRISPR loci,     -   the position of the CRISPR loci in the genome,     -   the number of repeat sequences, and/or     -   the nature and/or the size of the variable sequences.

1—Identification of the CRISPR Locus of the CNCM I-3689 Strain

The CNCM I-3689 strain was sequenced. The analysis of its genome using the ERGO™ software series made it possible to identify a single CRISPR locus in this strain. This locus, which is 3323 bp in size, is located 20 base pairs downstream of the ORF RDBK00370. Its genomic DNA sequence is represented by the sequence SEQ ID NO: 1. It is composed of a repeat unit (GTTTTCCCCGCACATGCGGGGGTGATCC; SEQ ID NO: 2) which is identical to that of the CRISPR locus of the ATCC 334 strain (Lactobacillus casei), and of 54 variable sequences (spacers).

2—Construction of PCR Amplification Primers Specific for the CNCM I-3689 Strain

Several pairs of oligonucleotide primers were defined on the basis of the variable sequences of the CRISPR locus of the CNCM I-3689 strain. These primers were tested by PCR amplification on several bacterial strains in order to verify their specificity for the CNCM I-3689 strain.

One pair of primers was retained:

-   -   primer OFF2486 (CTCAACAGGATAAGTGCCAC; SEQ ID NO: 3), located in         the 33^(rd) variable sequence of the CRISPR locus, at positions         2049-2068; TM=60° C.;     -   primer OFF2488 (GGTTGGCTGGGTTTAACGC; SEQ ID NO: 4), located in         the 37^(th) variable sequence of the CRISPR locus, at positions         2093-2110; TM=60° C.

The PCR conditions retained are the following:

-   -   Reaction mixture:

DNA: 1 μl dNTPs: 4 μl 10X buffer: 5 μl Primer OFF2486: 0.3 μl Primer OFF2488: 0.3 μl Ex tag ™ DNA polymerase: 0.2 μl Water: 39.2 μl

-   -   Cycles:

95° C. 5′ 95° C. 30″ 61° C. 30″ {close oversize brace} × 30 72° C. 45″ 72° C. 10′ 10° C.

The expected size of the PCR product from the CNCM I-3689 strain is 263 bp.

The pair of primers OFF2486/OFF2488 was tested on the CNCM I-3689 strain and on 18 other different strains of Lactobacillus casei, including the CNCM I-1518 and ATCC 334 strains. The CNCM I-1518 and ATCC 334 strains represent the negative controls since said primers cannot hybridize to the genomic DNA sequence of these strains under the PCR conditions described above.

The results are illustrated by the agarose gel electrophoresis of the PCR amplification products, presented in FIG. 3, where “121” represents the CNCM I-3689 strain, “001” represents the CNCM I-1518 strain, “S3” to “S18” represent, respectively, 16 different strains of Lactobacillus casei, and “SL” represents a molecular weight marker (SmartLadder; Eurogentec).

These results show that the pair of primers OFF2486/OFF2488 is indeed specific for the CNCM I-3689 strain, since PCR amplification products of expected size (i.e. approximately 260 bp) were obtained only for this strain. 

1-6. (canceled)
 7. A Lactobacillus paracasei subsp. paracasei strain having antimicrobial and immunomodulatory properties, characterized in that it is the strain deposited with the CNCM under number I-3689.
 8. A Lactobacillus paracasei subsp. paracasei strain having antimicrobial and immunomodulatory properties, obtained from a strain as claimed in claim 7 by mutagenesis or genetic transformation, wherein it has the antimicrobial and immunomodulatory properties of the CNCM I-3689 strain and/or has a CRISPR locus of sequence SEQ ID NO:
 1. 9. A method for obtaining a Lactobacillus paracasei subsp. paracasei strain having antimicrobial and/or immunomodulatory properties, comprising a step of mutagenesis or of genetic transformation of the CNCM I-3689 strain.
 10. A method for obtaining a cell fraction having antimicrobial and/or immunomodulatory properties, characterized in that said cell fraction is obtained from a Lactobacillus paracasei subsp. paracasei strain as claimed in claim
 7. 11. The method as claimed in claim 10, characterized in that said cell fraction is chosen from the group consisting of: a DNA preparation, a bacterial wall preparation, a culture supernatant and a fraction of said supernatant.
 12. A method for obtaining a composition having antimicrobial and immunomodulatory properties, comprising obtaining a cell fraction by means of a method as claimed in claim 9, and incorporating said fraction into said composition.
 13. The method as claimed in claim 12, wherein said product is a food product.
 14. A composition comprising a Lactobacillus paracasei subsp. paracasei strain as claimed in claim
 7. 15. A composition comprising a cell fraction obtained according to the method defined in claim
 10. 16. The composition as claimed in claim 14, wherein said composition is a food product.
 17. A method of treating a microbial infection in a subject in need thereof comprising administering an effective amount of the Lactobacillus paracasei subsp. paracasei strain as claimed in claim
 1. 18. A method of inhibiting inflammation in a subject in need thereof comprising administering an effective amount of the Lactobacillus paracasei subsp. paracasei strain as claimed in claim
 1. 